This proposal describes studies to probe structural changes in an enzyme during the course of its biochemical reaction. We propose to utilize rapid-freeze quench (RFQ) coupled with several spectroscopic techniques to probe the reaction of metallo-?-lactamase (M?L) L1 from Stenotrophomonas maltophilia as substrate binds and is converted into product. Specifically, ... In Specific aim #1, we propose to explore changes in metal site structure as a function of reaction coordinate, coupling rapid-freeze-quench (RFQ) techniques with conventional spectroscopies, including X-ray absorption spectroscopy (XAS), EPR and electron-nuclear double resonance (ENDOR), in the reaction of L1 with Zn and/or Co at the active site with a ?-lactam substrate. In Specific aim #2, we will examine solution dynamics of a position-conserved flexible loop that covers the metal site during turnover, through CW EPR studies of Co-containing derivatives of L1 incorporating a series of site-directed spin labels (SDSL);these studies will be extended into the time domain using RFQ-EPR to probe larger scale motions of the spin-labeled active site loop in the same Co-L1(SDSL) series. This proposal will offer a new strategy for the characterization of reactions catalyzed by Zn(II)- containing enzymes. More specific to the M?Ls, our long term goal is to identify structural/mechanism details common to all M?Ls, and in the future, we plan to apply the approaches, developed in this proposal, to other distinct M?Ls. The methodologies developed herein will easily lend themselves to the study of other metalloenzymes, regardless of the identity, oxidation level, or spin state of the metal. PUBLIC HEALTH RELEVANCE: The emergence of antibiotic resistance in the clinic has resulted in a biomedical crisis in which bacterial infections, once treated with inexpensive antibiotics, are now untreatable and cause a large number of deaths annually in the US and throughout the world. This proposal describes experiments to probe a metallo-?-lactamase, which causes resistance to penicillin and other ?-lactam containing antibiotics in the clinic, as it proceeds during catalysis. The information gleaned in these studies can be used to guide in the future development of M?L inhibitors.